CN108004338A - Detect the Primer composition and application and the product and detection SPF mouse pathogen methods using it of SPF mouse pathogens - Google Patents
Detect the Primer composition and application and the product and detection SPF mouse pathogen methods using it of SPF mouse pathogens Download PDFInfo
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Abstract
The present invention relates to technical field of biological, specifically, there is provided a kind of Primer composition of detection SPF mouse pathogens and application and application its product and detection SPF mouse pathogen methods.The Primer composition of detection SPF mouse pathogens provided by the invention can detect mycoplasma, mouse corynebacteria, Tyzzer organism and Pasteurella pneumotropica, the good no cross reaction of specificity at the same time；Reagent provided by the present invention for detecting SPF mouse pathogens detects quick, high sensitivity, high specificity；Can be with four kinds of pathogens of one-time detection provided by the present invention for the kit for detecting SPF mouse pathogens, detection is easy to operate, ensure that testing result is accurate, while reduces testing cost, may be directly applied to social production practice.The detection method of SPF mouse pathogen provided by the invention is easy to operate, as a result accurately, quick multiple detection method is provided for the detection research that SPF animal pathogens carry.
Description
Technical field
The present invention relates to technical field of biological, in particular to a kind of primer sets of detection SPF mouse pathogens
Compound and application and the product and detection SPF mouse pathogen methods using it.
Background technology
Experimental animal is basis and the supporting condition of life science, is that medical teaching and biomedical research are indispensable
Few experiment material, and the basis of medicine production detection and new drug research.Tremendous development and life section with social economy
Advancing by leaps and bounds for research is learned, Animal Science cause and occupational health and safety are increasingly valued by the people.Experiment is dynamic
The quality of thing directly affects the success or failure of experiment effect and research topic, determines the repeatability, feasibility and science of achievement in research
Property.Particularly zoonosis, or even directly threaten the health and lives of raising and laboratory staff safety.Periodically to experiment
It is the inspection to letting animals feed environmental facility and management final result that animal, which carries out microbiological quality monitoring, is to ensure that experiment is dynamic
The effective measures of amount of substance.
According to national standard, by the controlling extent of microorganism and parasite, the microbial standard of experimental animal is divided into
Four regular grade animal, cleaning grade animal, specific pathogen free animal (SPF) and germfree animal grades.SPF animals, are international
The experimental animal of upper the recognized standard rank, and whether experimental animal reaches SPF ranks, its important evaluation means is exactly to test
Animal microorganism, parasite quality control.It is to letting animals feed environment that microbiological quality monitoring is periodically carried out to experimental animal
Facility and the inspection for managing final result, are the effective measures for ensureing Quality of Experimental Animals.
According to National Standard of the People's Republic of China GB 14922.2-2011《Experimental animal microbiology grade and prison
Survey》, SPF mouse detection of pathogens project totally 15, detection project and must require 8 of feminine gender in detection of pathogens project,
Including mycoplasma, mouse corynebacteria, Tyzzer organism and Pasteurella pneumotropica.Mouse is modern medicine study and life
One of most common model animal of scientific research, ICR, KM, C57BL, BALB/c and BALB/c-nu be China's current needs amount most
Big several mouse species, SPF mouse are the experiment mices of the recognized standard rank in the world, to SPF mouse pathogen nucleic acid
Detection has very big necessity.
PCR or quantitative fluorescent PCR fast-bacteria-detection method are used at present, the majority of research is single or the inspection of two bacteriums
Survey, based on pure scientific research purpose.A variety of detection of pathogens of these methods such as application experiment animal, because project is more, each self-contained one
A detection architecture, parameter, condition are different, directly apply to social production practice, poor operability, and multi-pass operation cost
It is very high.A kind of quick, accurate, highly sensitive, high-throughout modern detecting is found, applies to the monitoring of experimental animal infectious disease
Work, is of great significance the quality and classification standard for ensureing experimental animal.
In view of this, it is special to propose the present invention.
The content of the invention
The first object of the present invention is to provide a kind of Primer composition of detection SPF mouse pathogens, and of the invention
In providing application of the above-mentioned Primer composition in SPF mouse pathogens are detected, the third object of the present invention is two purposes
Above-mentioned Primer composition is provided and is preparing the application in being used to detect the product of SPF mouse pathogens, the fourth object of the present invention
It is to provide a kind of reagent for being used to detect SPF mouse pathogens, the fifth object of the present invention is to provide a kind of for detecting
The kit of SPF mouse pathogens, the sixth object of the present invention be to provide a kind of detection method of SPF mouse pathogen, with
Alleviate and want whether carry mycoplasma, mouse corynebacteria, Tyzzer organism and thermophilic lung Pasteur in detection sample in the prior art
The problem of needing to detect one by one according to different experiments step, system and condition one by one during bacillus.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
The present invention provides a kind of Primer composition of detection SPF mouse pathogens, the Primer composition includes being used for
The primer pair of Tyzzer organism is detected, for detecting the primer pair of Pasteurella pneumotropica, for detecting drawing for mouse corynebacteria
Thing pair and the primer pair for detecting mycoplasma.
Further, the primer sequence of the primer pair for detecting Tyzzer organism is：
Sense primer-FP：5’-TACACTGGGATAACATCGAGAAATC-3’(SEQ ID NO.1)；
Anti-sense primer-RP：5’-ACCAACTAGCTAATCAGACGCGGGC-3’(SEQ ID NO.2)；
The primer sequence of the primer pair for detecting Pasteurella pneumotropica is：
Sense primer-FP：5’-TATGGAGGGGGATAACTGCGGGAAA-3’(SEQ ID NO.3)；
Anti-sense primer-RP：5’-AGACCAGCTAGAGATCGACGGCTTG-3’(SEQ ID NO.4)；
The primer sequence of the primer pair for detecting mouse corynebacteria is：
Sense primer-FP：5’-TAGTGTGTGTGGTGGAAAGTTTTTT-3’(SEQ ID NO.5)；
Anti-sense primer-RP：5’-GGCCGTATCTCAGTCCCAATGTGGC-3’(SEQ ID NO.6)；
The primer sequence of the primer pair for detecting mycoplasma is：
Sense primer-FP：5’-TGTGGTAGGGAGTTTTGGAATTTCA-3’(SEQ ID NO.7)；
Anti-sense primer-RP：5’-AGTATCTATCGTTTACGGTGTGGAC-3’(SEQ ID NO.8).
Present invention also offers application of the above-mentioned Primer composition in SPF mouse pathogens are detected.
Present invention also offers above-mentioned Primer composition to prepare for detecting answering in the product of SPF mouse pathogens
With.
Further, the product is reagent or kit.
Present invention also offers a kind of reagent for being used to detect SPF mouse pathogens, including above-mentioned Primer composition.
Present invention also offers a kind of kit for being used to detect SPF mouse pathogens, including above-mentioned Primer composition
Or reagent.
Present invention also offers a kind of detection method of SPF mouse pathogen, using above-mentioned Primer composition or reagent
Or kit.
Further, the described method includes：Using the Primer composition as primer, using the DNA of sample to be tested as template, into
The reaction of row fluorescent quantitative PCR is identified.
Further, the concentration of every kind of primer in the reaction system is 0.2pmol/ μ l in the Primer composition.
Compared with prior art, beneficial effects of the present invention are：
The Primer composition of detection SPF mouse pathogens provided by the invention can detect mycoplasma, mouse rod-like stem at the same time
Bacterium, Tyzzer organism and Pasteurella pneumotropica, the good no cross reaction of specificity；It is small provided by the present invention for detection SPF
The reagent of mouse pathogen includes Primer composition provided by the invention, and detection is quick, high sensitivity, high specificity；The present invention carries
The kit for being used to detect SPF mouse pathogens supplied once while can detect whether have Tyzzer organism in sample, thermophilic lung
P pestic, mouse corynebacteria and mycoplasma, kit detection is easy to operate, detects specific good, high sensitivity, ensures
Testing result is accurate, while reduces testing cost, may be directly applied to social production practice.SPF provided by the invention
The detection method of mouse pathogen is easy to operate, and detection speed significantly improves, and detection specificity is good, as a result accurately, reduces at the same time
Time cost and cost of labor, save resource, in SPF mouse pathogen must examine eight projects, can detect four knots at the same time
Fruit, quick multiple detection method is provided for the detection research that SPF animal pathogens carry.
Brief description of the drawings
Fig. 1 is amplification of the reaction system of the offer of the embodiment of the present invention 3 to Pasteurella pneumotropica DNA difference extension rates
Result figure；
Fig. 2 is amplification of the reaction system to mouse corynebacteria DNA difference extension rates of the offer of the embodiment of the present invention 3
Figure；
Fig. 3 is amplification of the reaction system to Tyzzer organism DNA difference extension rates of the offer of the embodiment of the present invention 3
Figure；
Fig. 4 is amplification figure of the reaction system to mycoplasma DNA difference extension rates of the offer of the embodiment of the present invention 3；
Fig. 5 A are that the reaction system that the embodiment of the present invention 3 provides is special to four kinds of pathogen quantitative fluorescent PCR joint-detections
Property analysis result schematic diagram；
Fig. 5 B are that the reaction system that the embodiment of the present invention 3 provides quickly detects four kinds of pathogen quantitative fluorescent PCR joints
Specific melting curve schematic diagram；
Fig. 6 A are that the specific analysis result of the SPF mouse pathogen carrying model sample detection of the embodiment of the present invention 5 is shown
It is intended to；
Fig. 6 B are that the specific dissolution curve of the SPF mouse pathogen carrying model sample detection of the embodiment of the present invention 5 shows
It is intended to.
Embodiment
Technical scheme is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation
Example is part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill
Personnel's all other embodiments obtained without making creative work, belong to the scope of protection of the invention.
The present invention provides a kind of Primer composition of detection SPF mouse pathogens, the Primer composition includes being used for
The primer pair of Tyzzer organism is detected, for detecting the primer pair of Pasteurella pneumotropica, for detecting drawing for mouse corynebacteria
Thing pair and the primer pair for detecting mycoplasma.
Further, the primer pair is：
Present invention also offers application of the above-mentioned Primer composition in SPF mouse pathogens are detected.
Present invention also offers above-mentioned Primer composition to prepare for detecting answering in the product of SPF mouse pathogens
With.Further, the product is reagent or kit.
Present invention also offers a kind of reagent for being used to detect SPF mouse pathogens, including above-mentioned Primer composition.Inspection
Survey quick, high sensitivity, high specificity.
Present invention also offers a kind of kit for being used to detect SPF mouse pathogens, including above-mentioned Primer composition
Or reagent.
The kit of above-mentioned detection SPF mouse pathogens further includes quantitative fluorescent PCR premix system and ddH2O.Fluorescence is determined
PCR premix system, that is, UItraSYBR Mixture are measured, including：Deoxyribonucleoside triphosphate, MgCl2, archaeal dna polymerase
And the PCR buffer solutions containing SYBR Green I and ROX dyestuffs.Quantitative fluorescent PCR premix system can be obtained directly in outsourcing.
ddH2O is distilled water, is one kind of redistilled water.
The kit of detection SPF mouse pathogens provided by the invention once can detect in sample whether have Tai Ze at the same time
Pathogen, Pasteurella pneumotropica, mouse corynebacteria and mycoplasma, kit detection is easy to operate, and detection specificity is good, spirit
Sensitivity is high, ensure that testing result is accurate, while reduces testing cost, may be directly applied to social production practice.
Present invention also offers a kind of detection method of SPF mouse pathogen, using above-mentioned Primer composition or reagent
Or kit.
Further, the described method includes：Using the Primer composition as primer, using the DNA of sample to be tested as template, into
The reaction of row fluorescent quantitative PCR is identified.
Wherein reaction system is：
Quantitative fluorescent PCR response procedures are：95 DEG C of denaturation 10min；95 DEG C of annealing 15s；60 DEG C of extension 1min, carry out 40
Circulation.
Further, the concentration of every kind of primer in the reaction system is 0.2pmol/ μ l in the Primer composition.
The detection method of SPF mouse pathogen provided by the invention is easy to operate, and detection speed significantly improves, and detection is special
Property it is good, as a result accurately, while reduce time cost and cost of labor, save resource, must examine eight projects in SPF mouse pathogen
In, four can be detected at the same time as a result, providing quick multiple detection method for the detection research that SPF animal pathogens carry.
In order to illustrate more clearly of the present invention, with reference to preferred embodiment, the present invention is described further.
1 design of primers of embodiment
Mycoplasma, mouse corynebacteria, Tyzzer organism and thermophilic lung Pasteur bar are downloaded from the NCBI gene databases in the U.S.
The genome sequence of bacterium.By being designed after carrying out sequence analysis and sequence analysis to the target gene fragment to be checked of each pathogen
Specific primer, the best four pairs of primers of specificity are obtained by contrast screening, specifying information it is following (the in sequence table the 1st, 2,
3,4,5,6,7,8 sequences)：
The primer commission Shanghai Ying Jun bio tech ltd synthesis that the present invention uses.
2 reaction system of embodiment and condition optimizing
Quantitative fluorescent PCR premix system (i.e. 2 × UItraSYBR Mixture (With ROX)) is public for century by Beijing health
Department provides.
Various pathogenic bacteria detection fluorescent quantitation reaction system is all 50 μ l, wherein 2 × UltraSYBR Mixture (With
ROX) 25 μ l, upstream and each 1 μ l of anti-sense primer, template DNA 5 μ l, ddH2O water complements to 50 μ l.
As a result judge：Fluoroscopic examination Mode S YBR is selected, the adjustment of fluorescence baseline takes the fluorescence signal of 3-15 circulation to be averaged
Value, threshold value setting are just above the peak of normal negative controls with threshold line, and sample is in typical amplification curve and in good
It is the positive that good logarithm, which increases, without typical amplification curve, is judged as feminine gender.
Optimized, quantitative fluorescent PCR reaction condition is as follows：95 DEG C of 10min denaturation；95 DEG C of 15s annealing；60 DEG C of 1min prolong
Stretch, then carry out fluoroscopic examination；Denaturation, annealing and extension carry out 40 circulations altogether.Melting curve analysis condition is：95 DEG C of 15s,
Fluoroscopic examination, 60 DEG C of 15s are carried out after 60 DEG C of 1min, 95 DEG C of 15s.
Using same concentrations positive nucleic acid in the reaction system of template, to be detected using different primers concentration, according to
It is 0.2pmol/ μ l that minimum Ct values and highest fluorescence intensity value added (Δ Rn), which select optimal primer concentration,.
3 sensitivity and specificity of embodiment detect
(a) strain to be tested sample DNA：Mycoplasma, mouse corynebacteria, the DNA of Pasteurella pneumotropica bacterial strain cure for Hangzhou
Institute preserves bacterial strain extract, and the DNA of Tyzzer organism is by Zhejiang Medical research institute Experimental Animal Center teacher Dai Fangwei
Give.Above-mentioned DNA extracts are diluted to 50ng/ul, as template mother liquor, on this basis, carry out 10-1、10-2、10-3、
10-4、10-5、10-6、10-7、10-8With 10-9Gradient dilution, obtains the DNA of various pathogenic bacteria various concentrations as subsequent PCR amplification
Template；
(b) using each gradient DNA as template, mixed with four pairs of specific primers and be configured to four reaction systems respectively and join
Close PCR detections：Four pairs of specific primers, PCR preparations, ddH2O；
(c) reaction product is placed in quantitative PCR apparatus and carries out fluoroscopic examination；Selection fluoroscopic examination pattern is SYBR fluorescence, baseline
Adjustment takes the fluorescence signal of 3~15 circulations, and the peak given threshold line of normal negative control is just above with threshold line；
PCR reaction fluorescence growth curves exceed threshold line, and increase in good logarithm, and it is good to embody detection architecture specificity.
The final concentration of each component is as follows in PCR reaction systems in the step (b)：
PCR reaction conditions are as follows in the step (b)：95 DEG C of 10min denaturation；95 DEG C of 15s annealing；60 DEG C of 1min extensions,
Then fluoroscopic examination is carried out；Denaturation, annealing and extension carry out 40 circulations altogether.
Experimental result such as Fig. 1 is amplification of the reaction system to Pasteurella pneumotropica DNA difference extension rates；Fig. 2
Amplification for reaction system to mouse corynebacteria DNA difference extension rates；Fig. 3 is reaction system to Tyzzer organism DNA
The amplification of different extension rates；Fig. 4 is amplification of the reaction system to mycoplasma DNA difference extension rates.
Pasteurella pneumotropica, mouse corynebacteria, Tyzzer organism, the examination of mycoplasma difference dilution factor DNA quantitative fluorescent PCRs
It is as shown in the table to test Ct values.
Specific test：
With Tyzzer organism, Pasteurella pneumotropica, the genomic DNA of mouse corynebacteria and mycoplasma is template, is used
Quantitative fluorescent PCR joint quick determination method detects the spy of above-mentioned four kinds of pathogen verification methods under the conditions of PCR amplification is optimized
The opposite sex, as a result as fig. 5 a and fig. 5b, it can be seen that the quantitative fluorescent PCR combined rapid detection reagent kit established of the present invention and
Detection method all has Tyzzer organism, mouse corynebacteria, mycoplasma and thermophilic lung Pasteur bar preferable specificity, to four kinds
The genomic DNA of cause of disease, which is detected, displays that positive reaction.And no cross reaction is detected between pathogen.
In Fig. 5 A：Ordinate expression correction of fluorescence intensity represents the circulation of fluorescent quantitative PCR to numerical value, abscissa
Number；Correction of fluorescence intensity equal to 0.100 horizontal line is threshold line to numerical value in figure, and curve 1-4 represents reaction system to safe pool disease
Substance, mouse corynebacteria, mycoplasma and Pasteurella pneumotropica detection of nucleic acids amplification, curve 5 represent the core of negative control
Sour augmentation detection result.(i.e. curve 1-4 is respectively in the reading of horizontal line Y crosspoints to reach the required period of threshold line for Ct values
Number)；
In Fig. 5 B figures：Ordinate expression correction of fluorescence intensity represents temperature to numerical value, abscissa；Curve 6-9 represents safe
Damp pathogen, mouse corynebacteria, the melting curve analysis result of mycoplasma and Pasteurella pneumotropica amplified production.10 table of curve
Show the nucleic acid solubility curve analysis result of negative control.
Sensitivity tests：
From the fluorescence quantitative PCR detection result of upper table, the reaction system experiment effect of design construction meets expection, energy
Specifically, sensitive, joint-detection experimental animal must examine pathogen infection Tyzzer organism, mouse corynebacteria, mycoplasma and thermophilic lung bar
The DNA of this moral bar.Calculated with Pasteurella pneumotropica fluorescent quantitative PCR result data instance, the detection of project team's structure design
System, the detection copy number to Pasteurella pneumotropica at 10 or so, i.e., 10 or so can be by sensitive, special containing bacteria concentration
Different amplification.Circular is as follows：
Method through DNA concentration and Q-PCR results presumption Bacteria Detection sensitivity copy numbers：Pasteurella pneumotropica
The DNA sizes of 2.43Mb.The base number N of one genome is exactly 2 × 2.43 × 10^6.Molecular weight is exactly 324.5 × 2 × 2.43
×10^6=1.58 × 10^9；The template of reaction system 1 μ l is added equivalent to having 333.5ngDNA, i.e. 3.335 × 10^-7g.This
Sample, genome molal quantity, i.e. bacterium molal quantity are exactly 3.335 × 10^-7, divided by 1.58 × 10^9=2.12 × 10-16Mol (samples
The molal quantity of product DNA).Multiplied by with 2.12 × 10^ of Avgadro constant-16×6.02×10^23=1.3 × 10^8Copy.About
Equal to 10^8Copy.The Ct value general controls of qPCR are relatively good in 15-35, in this experiment, Pasteurella pneumotropica DNA dilutions times
Number is 10-6When, preferable Ct values still can be obtained, infer system detection sensitivity in bacterial copy number 10 or so.
Further use experimental verification calculated value：Mouse corynebacteria, mycoplasma and Pasteurella pneumotropica are tested
Confirmation is tested.Mouse corynebacteria, mycoplasma and Pasteurella pneumotropica do dilution gradient：105、104、103、102、101/ ml is each
Reaction tube, and by using the DNA of bacteria extracts kit of precious bioengineering Co., Ltd, by kit specification the method
Extract the genomic DNA of various pathogenic bacteria.By being used as joint fluorescent quantitation using the DNA extracts of different dilution gradient pathogens
The template of PCR detections.It is detected through fluorescence quantifying PCR method, the results show combines the cause of disease of fluorescent quantitative PCR detection method
Bacterium detection sensitivity reaches 101/ml。
Since Tyzzer organism cannot be grown on no living cells synthetic medium, and extreme anaerobism, pure culture bacterial strain obtain
Difficulty is taken, so Tyzzer organism sensitivity Detection carries out above-mentioned sensitivity technique with the dilution of extract DNA ladder degree, it is dense through DNA
Degree and Q-PCR results presumption Bacteria Detection sensitivity copy numbers are 10 or so.
4 repetitive test of embodiment
Various concentrations DNA moulds using the joint fluorescent quantitative PCR detection method that the present invention is established to various pathogenic bacteria
Version is detected, and reaction system makees 5 repetition detections as described in Example 2, to the sample of each concentration, as a result different nucleic acid
The respective detection Ct value standard deviations of concentration have preferable repeatability between 0.05~0.15.
Embodiment 5SPF mouse pathogen carries the detection of pathogens application of model sample
The experiment of viable bacteria infecting mouse, Tyzzer organism inspection have been done to mouse corynebacteria, mycoplasma and Pasteurella pneumotropica
Extract DNA is used in survey.Model is carried by establishing SPF mouse pathogen, with various pathogenic bacteria infecting mouse model.Gather SPF
The body fluid and tissue sample of infecting mouse directly extracts DNA of bacteria, the joint fluorescent PCR detection side invented afterwards using this research
Method is to Tyzzer organism, mouse corynebacteria, mycoplasma and thermophilic lung Pasteur bar DNA extracts are detected respectively and application simulation
Experiment, experimental result show that the detection method of the present invention can carry out purpose pathogen DNA extracts effectively to detect (Fig. 6 A
With Fig. 6 B).The results show this method is detected the genomic DNA of four kinds of pathogens the special positive reaction of display, and disease
There is no cross reaction between opportunistic pathogen.
In Fig. 6 A：Ordinate expression correction of fluorescence intensity represents the circulation of fluorescent quantitative PCR to numerical value, abscissa
Number；Correction of fluorescence intensity equal to 0.100 horizontal line is threshold line to numerical value in figure, and curve 1-4 represents reaction system to safe pool disease
Substance, mouse corynebacteria, mycoplasma and Pasteurella pneumotropica detection of nucleic acids amplification, curve 5 represent the core of negative control
Sour augmentation detection result.(i.e. curve 1-4 is respectively in the reading of horizontal line Y crosspoints to reach the required period of threshold line for Ct values
Number)；
In Fig. 6 B figures：Ordinate expression correction of fluorescence intensity represents temperature to numerical value, abscissa；Curve 6-9 represents safe
Damp pathogen, mouse corynebacteria, the melting curve analysis result of mycoplasma and Pasteurella pneumotropica amplified production.10 table of curve
Show the nucleic acid solubility curve analysis result of negative control.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, it will be understood by those of ordinary skill in the art that：Its according to
Can so modify to the technical solution described in foregoing embodiments, either to which part or all technical characteristic into
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
SEQUENCE LISTING
<110>Hangzhou Medical institute
<120>Detect Primer composition and its application and the product using it and the detection SPF mouse of SPF mouse pathogens
The method of pathogen
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
tacactggga taacatcgag aaatc 25
<210> 2
<211> 25
<212> DNA
<213>Artificial sequence
<400> 2
accaactagc taatcagacg cgggc 25
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
tatggagggg gataactgcg ggaaa 25
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence
<400> 4
agaccagcta gagatcgacg gcttg 25
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence
<400> 5
tagtgtgtgt ggtggaaagt ttttt 25
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence
<400> 6
ggccgtatct cagtcccaat gtggc 25
<210> 7
<211> 25
<212> DNA
<213>Artificial sequence
<400> 7
tgtggtaggg agttttggaa tttca 25
<210> 8
<211> 25
<212> DNA
<213>Artificial sequence
<400> 8
agtatctatc gtttacggtg tggac 25
Claims (10)
1. a kind of Primer composition of detection SPF mouse pathogens, it is characterised in that the Primer composition includes being used to detect
The primer pair of Tyzzer organism, for detecting the primer pair of Pasteurella pneumotropica, for detecting the primer pair of mouse corynebacteria
With the primer pair for detecting mycoplasma.
2. Primer composition according to claim 1, it is characterised in that
The primer sequence of the primer pair for detecting Tyzzer organism is：
Sense primer-FP：5’-TACACTGGGATAACATCGAGAAATC-3’(SEQ ID NO.1)；
Anti-sense primer-RP：5’-ACCAACTAGCTAATCAGACGCGGGC-3’(SEQ ID NO.2)；
The primer sequence of the primer pair for detecting Pasteurella pneumotropica is：
Sense primer-FP：5’-TATGGAGGGGGATAACTGCGGGAAA-3’(SEQ ID NO.3)；
Anti-sense primer-RP：5’-AGACCAGCTAGAGATCGACGGCTTG-3’(SEQ ID NO.4)；
The primer sequence of the primer pair for detecting mouse corynebacteria is：
Sense primer-FP：5’-TAGTGTGTGTGGTGGAAAGTTTTTT-3’(SEQ ID NO.5)；
Anti-sense primer-RP：5’-GGCCGTATCTCAGTCCCAATGTGGC-3’(SEQ ID NO.6)；
The primer sequence of the primer pair for detecting mycoplasma is：
Sense primer-FP：5’-TGTGGTAGGGAGTTTTGGAATTTCA-3’(SEQ ID NO.7)；
Anti-sense primer-RP：5’-AGTATCTATCGTTTACGGTGTGGAC-3’(SEQ ID NO.8).
3. application of the Primer composition as claimed in claim 1 or 2 in SPF mouse pathogens are detected.
4. Primer composition as claimed in claim 1 or 2 is being prepared for detecting answering in the product of SPF mouse pathogens
With.
5. application according to claim 4, it is characterised in that the product is reagent or kit.
6. a kind of reagent for being used to detect SPF mouse pathogens, it is characterised in that including the primer sets described in claim 1 or 2
Compound.
7. a kind of kit for being used to detect SPF mouse pathogens, it is characterised in that including the primer described in claim 1 or 2
Reagent described in composition or claim 6.
8. a kind of detection method of SPF mouse pathogen, it is characterised in that using the Primer composition described in claim 1 or 2
Or the reagent described in claim 6 or the kit described in claim 7.
9. detection method according to claim 8, it is characterised in that the described method includes：Using the Primer composition as
Primer, using the DNA of sample to be tested as template, carries out fluorescent quantitative PCR reaction and is identified.
10. detection method according to claim 9, it is characterised in that every kind of primer is reacting in the Primer composition
Concentration in system is 0.2pmol/ μ l.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005034121A (en) * | 2003-07-15 | 2005-02-10 | Nagasaki Prefecture | En bloc detection method for bacterium causing food poisoning and reagent therefor |
| KR20090027875A (en) * | 2007-09-13 | 2009-03-18 | 주식회사 코젠바이오텍 | Method of detecting pathogens responsible for food poisoning and fast detection kit |
| CN103740822A (en) * | 2013-12-31 | 2014-04-23 | 浙江医学高等专科学校 | Fluorescence quantitative PCR (polymerase chain reaction) combined rapid detection kit and method for SPF (specific pathogen free) mice pathogenic bacteria nucleic acid |
| CN106520986A (en) * | 2016-12-06 | 2017-03-22 | 河北医科大学 | Quintuple PCR (polymerase chain reaction) detection method capable of detecting multiple pathogens simultaneously |
| CN106755589A (en) * | 2017-01-10 | 2017-05-31 | 广东省实验动物监测所 | Five kinds of primer sets of pathogen of rat, kit and Multiple immunizations fluorescence analysis method are detected simultaneously |
| CN107236793A (en) * | 2017-05-31 | 2017-10-10 | 广东省妇幼保健院 | A kind of Corynebacterium sp. bacteria and kirschner corynebacteria multiple fluorescence quantitative PCR primer, probe, kit and detection method |
| CN107338315A (en) * | 2017-08-15 | 2017-11-10 | 中国人民解放军总医院 | Kit for 15 kinds of pneumonia pathogenic bacteria quick detections |
-
2017
- 2017-12-22 CN CN201711416698.5A patent/CN108004338B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005034121A (en) * | 2003-07-15 | 2005-02-10 | Nagasaki Prefecture | En bloc detection method for bacterium causing food poisoning and reagent therefor |
| KR20090027875A (en) * | 2007-09-13 | 2009-03-18 | 주식회사 코젠바이오텍 | Method of detecting pathogens responsible for food poisoning and fast detection kit |
| CN103740822A (en) * | 2013-12-31 | 2014-04-23 | 浙江医学高等专科学校 | Fluorescence quantitative PCR (polymerase chain reaction) combined rapid detection kit and method for SPF (specific pathogen free) mice pathogenic bacteria nucleic acid |
| CN106520986A (en) * | 2016-12-06 | 2017-03-22 | 河北医科大学 | Quintuple PCR (polymerase chain reaction) detection method capable of detecting multiple pathogens simultaneously |
| CN106755589A (en) * | 2017-01-10 | 2017-05-31 | 广东省实验动物监测所 | Five kinds of primer sets of pathogen of rat, kit and Multiple immunizations fluorescence analysis method are detected simultaneously |
| CN107236793A (en) * | 2017-05-31 | 2017-10-10 | 广东省妇幼保健院 | A kind of Corynebacterium sp. bacteria and kirschner corynebacteria multiple fluorescence quantitative PCR primer, probe, kit and detection method |
| CN107338315A (en) * | 2017-08-15 | 2017-11-10 | 中国人民解放军总医院 | Kit for 15 kinds of pneumonia pathogenic bacteria quick detections |
Non-Patent Citations (4)
| Title |
|---|
| 唐连飞等: "鼠棒状杆菌 PCR 检测方法的建立及其应用", 《中国比较医学杂志》 * |
| 王晶: "屏障系统微生物气溶胶监测及SPF级实验动物微生物多重PCR分析", 《中国优秀硕士学位论文全文数据库》 * |
| 王鹏飞: "实验动物金黄色葡萄球菌、绿脓杆菌和肺支原体多重PCR检测方法的建立与初步应用", 《中国优秀硕士学位论文全文数据库》 * |
| 祝岩波等: "实验动物泰泽氏菌、肺支原体、兔出血症病毒检测方法的评价", 《实验动物科学》 * |
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